The ability to convert human skin cells to induced pluripotent stem cells (IPSCs) represents a seminal break-through in stem cell biology. This advance effectively circumvents the problem of immune rejection because the patient’s own skin cells can be used to produce iPSCs. This exciting technology could accelerate treatments for a number of presently incurable diseases. However, a paramount unanswered question is whether these cells or their derivatives are truly safe for administration. Specifically, it is unknown whether the integrity of the iPSC genome is maintained during the tissue culture steps required to generate, maintain, expand and differentiate iPSCs. Every cell contains roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of the human genome. This contrasts with the fact that the roughly 21,000 human genes occupy only 1.5% of genome. While many of these retroelements have been permanently silenced during evolution, many others remain active and capable of replicating and moving to new chromosomal locations potentially producing disease-causing mutations or cancer. Somatic cells limit the jumping of these mobile genetic elements (retrotransposition) chiefly by methylating the DNA in and around these elements. Strikingly, the process of converting a skin cell to an iPSC results in a profound loss of DNA methylation potentially opening the door for high level retroelement activity that could corrupt genomic integrity. These insertions can disrupt key genes, create double strand DNA breaks or lead later to loss of large sections of DNA. Whether retroelement activity contributes to the fact that only 0.01% of skin cells are successfully reprogrammed to iPSCs is unknown. Thus, key questions regarding the safety of these cells remains. We now propose to determine the level of retroelement retrotransposition occurring in iPSCs and hESCs and to develop potentially safer ways to generate and maintain iPSCs in culture by blocking a key retroelement enzyme. Further, we will assess whether differentiation of these cells triggers retroelement activity. Finally, we will explore potential additional cellular defenses brought into action to oppose these retroelements with the goal of further enhancing these defenses.

Statement of Benefit to California:

The use of pluripotent stem cells derived either from the inner cell mass of developing blastocysts or by reprogramming of skin cells holds great therapeutic promise. These cells could provide exciting new approaches for a number of incurable human diseases like Parkinson’s and Alzheimer’s disease, type 1 diabetes, and cardiac failure. However, a paramount unanswered question in the field is whether these cells can be used in a completely safe manner. One major threat that could undermine these exciting stem cell therapies is the appearance of genetic mutations during their generation, expansion or differentiation. Such mutations could be induced by mobile genetic elements. Every cell contains roughly 3 million mobile genetic retroelements that comprise up to 45% of the genome. Active retroelements are capable of reproducing themselves and then jumping to a new chromosomal location potentially causing devastating disease-causing mutations or cancer-promoting changes. In normal differentiated cells, the jumping of these retroelements is highly constrained by DNA methylation. However, when skin cells are reprogrammed to become induced pluriopotent stem cells (iPSCs), DNA methylation is essentially erased. Human embryonic stem cells (hESCs) also exhibit dynamic changes in DNA methylation characterized by rapid losses and gains. These events open the door for repeated waves of retroelement retrotransposition that could greatly undermine the genome integrity of these cells. A real gap in our understanding of iPSC biology is that the potential activity and damaging effects of these retroelements has not been explored. To determine if hESCs and iPSCs and their cellular progeny can be safely used in patients, we propose to study the expression of retroelement RNA, the frequency of physical jumping events, and the impact of potential cellular defensive mechanisms opposing these retroelements. Since stem cells will be differentiated in vitro prior to their use in patients, we will also study levels of retroelement jumping in cells induced to differentiate into the three germ cell layers, endoderm, mesoderm and ectoderm. Additionally, we propose to explore potentially safer ways to generate and maintain iPSCs in culture where the retrotransposition process is interrupted using an FDA-approved HIV antiviral drug. Such an approach could protect the genome of these cells during their culture and manipulation in the laboratory prior to infusion into patients. The results of these studies will have both important scientific and practical value for the future therapeutic use of stems cells. As such, we believe these studies will benefit the citizens of California certainly at a societal level and potentially at a personal level.

Progress Report:

Year 1

The use of human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells holds great therapeutic promise for a number of currently incurable human diseases. However, successful deployment of these cells requires successful negotiation of several “bottlenecks” some of which involve the safety of the derived cells for infusion. One important safety concern is that during the generation, expansion, and manipulation of these pluripotent cells, mutations may be introduced into the genome due to increased “jumping” of mobile genetic elements.
Every cell contains roughly 3 million “jumping genes” or endogenous retroelements that comprise up to 45% of the DNA present in the human genome. Fortunately, many of these retroelements have been permanently silenced during evolution by crippling mutations. Nevertheless, some remain active and capable of moving to new chromosomal locations potentially producing disease-causing mutations or cancer. More mature differentiated cells control retroelement movement (retrotransposition) chiefly by methylating the DNA comprising these retroelements. Strikingly, such DNA methylation is highly dynamic in hES cells because these cells must be able to differentiate into a wide spectrum of different cell types leading to tissue and organ generation. During reprogramming of skin cells to iPS cells, DNA methylation patterns are erased, potentially making iPS cells vulnerable to heightened retroelement activity. Our CIRM-sponsored work focuses on assessing endogenous retroelement activity as skin cells are reprogrammed into iPS cells. During the first year of the funding, we have shown that endogenous retroelements are very active in iPS cells and thus potentially endanger the genomic integrity of these cells. We are now exploring whether diminishing retroelement mobilization by treatment with specific drugs may provide a safer way to produce, culture and expand pluripotent cells.
In addition we are exploring several cellular defenses beyond DNA methylation that may counter retroelement retrotransposition in iPS cells (e.g., APOBECs, TREX-1, and the RNAi machinery). One goal is to assess whether one or more of these pathways is particularly active in iPS and hES cells and specifically used to control retroelement activity. Together, these studies are systematically addressing endogenous retroelement activity in pluripotent stem cells with an eye to limiting their activity and thus making the generation of these cells and their progeny safer.

Year 2

Human embryonic stem (hES) cells and induced pluripotent (iPS) cells hold great promise as regenerative therapies for a number of currently incurable human diseases. However, before these cells or their progeny can be used as infusion therapies, the safety of these cells must be confirmed. One important safety concern is that during the generation, expansion, and manipulation of these pluripotent cells, mutations may be introduced into their genomes as a result of the activation and “jumping” of endogenous mobile genetic elements. Human cells contain roughly 3 million endogenous retroelements that comprise slightly less than half of the DNA present in the entire genome. Although many of these retroelements have been permanently silenced due to mutations, many others remain retrotransposition-competent and are capable of producing RNA, converting this RNA back into DNA (reverse transcription) and inserting this DNA at new sites in the cellular genome. These new insertions expand the cell’s pool of DNA and potentially produce disease-causing mutations or cancer. In somatic cells, the expression of these retroelements is strongly repressed through DNA methylation. However, because of the dynamic changes in DNA methylation that occur during the generation of iPS cells, high level retroelement retrotransposition may be unleashed. Our CIRM-sponsored work focuses on assessing endogenous retroelement activity as skin cells are reprogrammed into iPS cells. In the last 6 months, we have demonstrated that endogenous retroelements jump during iPS cell reprogramming and that the iPS cells generated contain new retroelement insertions in their genomes. We are now creating a new method to monitor the retroelement retrotransposition more accurately during reprogramming. In addition we are exploring both small molecules and cellular defenses that may counter retroelement retrotransposition in iPS cells (e.g., DNA methylation, APOBECs, TREX-1, and the RNAi machinery). Together, our goal is to provide a safer way to produce, culture and expand pluripotent cells.

Year 3

Human embryonic stem (hES) cells and induced pluripotent (iPS) cells holds great promise as regenerative therapies for a number of currently incurable human diseases. However, before these cells or their progeny can be used as infusion therapies, the safety of these cells must be confirmed. One important safety concern is that during the generation, expansion, and manipulation of these pluripotent cells, mutations may be introduced into their genomes as a result of the activation and “jumping” endogenous mobile genetic elements. Human cells contain roughly 3 million endogenous retroelements that comprise slightly less than half of the DNA present in the entire genome. Although many of these retroelements have been permanently silenced due to mutations, many others remain retrotransposition-competent and are capable of producing RNA, converting this RNA back into DNA (reverse transcription) and inserting this DNA at new sites in the cellular genome. These new insertions expand the cell’s pool of DNA and potentially produce disease-causing mutations or cancer. In somatic cells, the expression of these retroelements is strongly repressed through DNA methylation. However, because of the dynamic changes in DNA methylation that occur during the generation of iPS cells, high level retroelement retrotransposition may be unleashed. Our CIRM-sponsored work focuses on assessing endogenous retroelement activity as skin cells are reprogrammed into iPS cells. In the last 6 months, We made significant progress by applying whole-genome sequencing to investigate the LINE-1 and Alu retrotransposition in iPSCs. We also demonstrated that endogenous retroelements jump when iPSCs were differentiated to hematopoietic stem cell progenitors.